Single Cell RNA-Sequencing of Skeletal Muscle in Dystrophic Mouse Models

Duchenne muscular dystrophy (DMD) is caused by an out-of-frame mutation in the DMD gene that results in the absence of a functional dystrophin protein, leading to a devastating and progressive lethal muscle-wasting disease. Genetic modifiers have been shown to increase disease severity in DMD mouse models as well as in human patients. Little is known about cellular heterogeneity in skeletal muscle as disease severity increases. To address this, we explored skeletal muscle-resident cell populations in healthy (wt-NSG), dystrophic (mdx-NSG), and severely dystrophic (mdxD2-NSG) mouse models utilizing scRNA-seq. We found an increased frequency of activated fibroblasts, activated fibro-adipogenic progenitor cells, and proinflammatory macrophages in dystrophic and severely dystrophic gastrocnemius muscles. Moreover, in endothelial cells we found an upregulation of extracellular matrix and platelet aggregation genes in dystrophic and severely dystrophic muscles, indicating endothelial cell functional impairment. In summary, this work extends the understanding of the severe nature of DMD, which should be considered when developing regenerative therapeutic avenues for DMD. Overall design: Gastrocnemius muscles were harvested from the right hindlimb of 8 weeks old wt-NSG, mdx-NSG, and mdxD2-NSG male mice each separately. The digested muscles were sorted for live cells, and 10X Chromium Single Cell 3' library construction was performed followed by Ilumina NovaSeq 6000 sequecning. Single cells were analyzed using R package Seurat V 3.1.5 for broad cell type identification (Butler et al., 2018). Further integration of all single cells from all mouse models was carried out using R package Harmony (Korsunsky et al., 2019). Cellular heterogenity between healthy (wt-NSG), dystrophic (mdx-NSG) and severly dystrophic (mdxD2-NSG) skeletal muscles were then investigated.